Analysis of HSA–PAA Complexation Using SEC-SAXS Combination: Unraveling Stoichiometry, Reversibility, and Interaction Specificity

Abstract

This work leverages the integration of size exclusion chromatography (SEC) with small-angle X-ray scattering (SAXS) to investigate the complex interactions between human serum albumin (HSA) and poly­(acrylic acid) (PAA). The SEC-SAXS approach is proven in this study to effectively eliminate aggregates, enhancing data quality and revealing intricate details of protein–polymer associations. Initial findings demonstrate that HSA maintains its native structure across pH 5–8 and that HSA shows no significant interaction with the neutral polyethylene glycol (PEG). This highlights the critical role of charge regulation and electrostatic forces in HSA–PAA complex formation previously reported and the specificity of this interaction. The study further reveals that the HSA–PAA complex stoichiometry is highly dependent on PAA size, with larger PAA chains forming more elongated structures. The binding stoichiometry is then shown to increase nonlinearly, suggesting a delicate balance between attractive HSA–PAA and repulsive HSA–HSA interactions. Notably, HSA–PAA complexes exhibit reversible behavior, dissociating at pH > 5 and in media devoid of PAA.

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